Preparative Gel Electrophoresis of Native Metalloproteins

by Bernd Kastenholz, David E. Garfin, and Jürgen Horst

We have developed a method termed “quantitative preparative native continuous polyacrylamide gel electrophoresis” (QPNC-PAGE) that is a high-resolution technique for separating proteins by isoelectric point. We demonstrate this method with conditions that have been used to isolate active, native metalloproteins and to resolve properly- and improperly-folded metal cofactor-containing proteins in complex protein mixtures [1-6].

QPNC-PAGE is based on Bio-Rad’s Model 491 Prep Cell, an instrument for preparative gel electrophoresis of all kinds. The procedure is done in an unmodified Prep Cell (Figure 1). Through use of a high-pH buffer, most proteins are negatively charged and migrate from the cathode to the anode during electrophoresis [1]. Although the pH value (pH 10) of the electrophoresis buffer is not physiological, protein isomers are continuously eluted at physiological pH (pH 8) [2].

Figure 1. Electrophoresis chamber Model 491 Prep Cell from Bio-Rad for isolating protein molecules in complex biological matrices. This figure is used with the permission of Bio-Rad Laboratories, Inc.

In order to ensure full polymerization, the polymerization reaction is allowed to go for 69 hr at room temperature. As a result, the prepared gel is homogeneous, mechanically stable, and free of monomers or radicals. The pore sizes of the prepared gel are very large and therefore sieving effects are minimal [3,4]. Separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) are not dissociated into apoproteins and metal cofactors under the conditions given here. We believe also that metalloproteins do not undergo significant conformational changes during QPNC-PAGE. Consequently, quantitative amounts of highly purified metalloproteins are isolated in a few specific PAGE fractions (Figure 2) [3,4].

Figure 2. Electropherogram showing the elution profile of cadmium-bearing proteins (Cd proteins) from Arabidopsis thaliana (inset; Wikipedia). High molecular-mass proteins were first obtained by size exclusion chromatography. The cadmium concentrations in each fraction were determined by GF-AAS (corrected base line) [4]. This figure is used with permission of Bentham Science Publishers Ltd.

Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and quantified by inductively coupled plasma mass spectrometry or by graphite furnace atomic absorption spectrometry (GF-AAS) as was done, for example, in the fractionation shown in Figure 2.

METHOD

Table 1. Recommended Equipment and Run Conditions

Model 491 Prep Cell
Power Pac 1000 - Constant Power: 5 W; Time: 8 hr
Model EP-1 Econo Pump - 1 mL/min; 5 mL/Fract.; 80 mL Prerun V; 480 ml Total V (Eluent)
Model 2110 Fraction Collector - 80 Fractions
Model EM-1 Econo UV Monitor - AUFS 1.0; Detection Wavelength 254 nm
Model 1327 Econo Recorder - Range: 100 mV; Chart Speed: 6 cm/hr
Model SV-3 Diverter Valve
Buffer Recirculation Pump - Flow Rate: 95 mL/min (Electrophoresis Buffer)

All instruments are from Bio-Rad Laboratories, Inc.

SOLUTIONS

-Stock Solutions

  • 200 mM Tris-HCl, 10 mM NaN3, pH 10.00 — Store at room temperature
  • 200 mM Tris-HCl, 10 mM NaN3, pH 8.00 — Store at room temperature
  • 40 % Acrylamide/Bis 2.67 % C — Store at 4°C
  • 10 % Ammonium Persulfate (APS) — Store at 4°C (freshly prepared)

-Electrophoresis Buffer

  • 20 mM Tris-HCl, 1 mM NaN3, pH 10.00 — Store at 4°C and degas before use
  • Upper Electrophoresis Chamber (Prep Cell): 500 mL Electrophoresis Buffer
  • Lower Electrophoresis Chamber (Prep Cell): 2000 mL Electrophoresis Buffer

-Eluent

  • 20 mM Tris-HCl, 1 mM NaN3, pH 8.00 — Store at 4°C and degas before use
  • Elution Chamber (Prep Cell): 700 mL Elution Buffer

GEL

-Separating Gel

  • Acrylamide 4% T Volume: 40 mL
  • ingredients:
  • 4 mL 40% Acrylamide/Bis 2.67% C
  • 4 mL 200 mM Tris-HCl, 10 mM NaN3, pH 10.00
  • 32 mL H2O
  • 200 µL 10% APS
  • 20 µL TEMED

Add TEMED and APS last. Gently swirl the beaker to mix, being careful not to generate bubbles. Pipette the solution to a level of 40 mm in the 28 mm ID gel tube assembly. Add 3 mL of 2-propanol. After 60 minutes of polymerization rinse the surface of the gel with electrophoresis buffer and then cover the gel surface with 4 mL electrophoresis buffer. Allow the polymerization reaction to proceed for 69 hr at RT. The heat generated during the polymerization processes is dissipated according to the Model 491 Prep Cell instruction manual [7]. There is no stacking gel.

APPLICATION

The structure-function relationships of isolated metalloproteins in brain, blood or other clinical samples are important because improperly folded metal proteins, for example, copper chaperones for superoxide dismutase (CCS) or superoxide dismutase (SOD), present in these biomatrices may be responsible for neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis. Active CCS or SOD molecules contribute to intracellular homeostatic control of metal ions in organisms and thus these biomolecules can balance pro-oxidative and anti-oxidative processes in the cytoplasm [3-5]. A combined procedure consisting of QPNC-PAGE, SEC, ICP-MS and solution NMR is an effective approach for elucidating the different structures of physiologically relevant metalloproteins in biofluids (e.g., urine, blood) of patients and probands. It is anticipated that the results obtained by this analytical process will contribute to an early diagnosis and therapy of several protein-misfolding diseases (Figure 3) [3-6].

Figure 3. Workflow schemes for analysis of metalloproteins and protein-protein interactions incorporating QPNC-PAGE [5]. Used with permission of Bentham Science Publishers Ltd.

REFERENCES

[1] Bernd Kastenholz (2004). "Preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE): An efficient method for isolating cadmium cofactors in biological systems". Analytical Letters 37: 657—665.

[2] Bernd Kastenholz (2006). "Comparison of the electrochemical behavior of the high molecular mass cadmium proteins in Arabidopsis thaliana and in vegetable plants on using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE)". Electroanalysis 18: 103—106.

[3] Bernd Kastenholz (2006). "Important contributions of a new quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) procedure for elucidating metal cofactor metabolisms in protein-misfolding diseases — a theory". Protein and Peptide Letters 13: 503—508.

[4] Bernd Kastenholz (2007). "New hope for the diagnosis and therapy of Alzheimer's disease". Protein and Peptide Letters 14: 389—393.

[5] Bernd Kastenholz; David E. Garfin (2009). "Medicinal plants: a natural chaperones source for treating neurological disorders". Protein and Peptide Letters 16: 116—120.

[6] Bernd Kastenholz; David E. Garfin; Jürgen Horst; Kerstin A. Nagel (2009). "Plant metal chaperones: a novel perspective in dementia therapy". Amyloid-Journal of Protein Folding Disorders 16: in press.

[7] Bio-Rad. “Model 491 Prep Cell, Instruction Manual, Rev C”. Bulletin # M1702925: 1-47.


Affiliation

  • Bernd Kastenholz1*, David E. Garfin2, and Jürgen Horst3
  • 1Forschungszentrum Jülich GmbH, Phytosphere Institute (ICG-3), 52425 Jülich, Germany, e-mail: b.kastenholz@fz-juelich.de
  • 2American Electrophoresis Society, 1202 Ann Street, Madison, WI 53713, USA, e-mail: degarfin@sonic.net
  • 3 Westfälische Wilhelms-Universität Münster, Institut für Humangenetik, 48149 Münster, Germany, e-mail: horstj@uni-muenster.de

*Corresponding author